Regulation Of Hematopoietic Stem Cells Research Articles

A Genetic Disorder Reveals a Hematopoietic Stem Cell Regulatory Network Co-Opted in Leukemia

The molecular regulation of human hematopoietic stem cell (HSC) self-renewal and maintenance is of substantial interest, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. To better discern in vivo HSC function in humans, we have studied a rare genetic disorder due to MECOM haploinsufficiency that is characterized by neonatal aplastic anemia with an early-onset absence of HSCs in vivo.To establish a faithful model of MECOM haploinsufficiency, we performed CRISPR/Cas9 editing of MECOM in human hematopoietic stem and progenitor cells (HSPCs) and achieved predominantly heterozygous editing in phenotypic long-term (LT)-HSCs, as substantiated through both bulk and single cell assessments. Following MECOM editing, HSPCs showed a significant reduction in the number of phenotypic LT-HSCs as well as multipotential progenitor colonies in vitro, and had impaired engraftment following xenotransplantation into immunodeficient and Kit mutant mice. Next, we sought to use this model of MECOM haploinsufficiency to define the regulatory networks driven by MECOM that are critical for HSC maintenance. We therefore performed single-cell RNA sequencing on several thousand phenotypic LT-HSCs following MECOM editing and identified a list of 724 subtly but significantly differentially expressed genes compared to controls, including 322 genes that are downregulated after MECOM perturbation. Given the profound phenotypic effects associated with loss of MECOM and dysregulation of these gene sets, we sought to identify other cooperating factors that control the MECOM dependent gene network which underlies HSC self-renewal. To do so, we used integrative genomic approaches involving accessible chromatin and gene expression correlations, as well as long range chromatin interaction data across human hematopoiesis, to comprehensively define associations between genes and putative regulatory elements. Inspection of the nominated cis-regulatory elements controlling genes impacted by MECOM perturbation revealed significant enrichment for motifs and chromatin occupancy by several key cooperating transcription factors, including RUNX1, FLI1, and GATA2.In addition to the identification of cooperating transcription factors, we also discovered a strong binding motif enrichment for, and chromatin occupancy by, CTCF. CTCF is crucial to enable differentiation of LT-HSCs, and we found that MECOM regulated genes frequently had associated cis-regulatory elements that were occupied by CTCF. Moreover, these occupied cis-elements became more highly enriched for binding during hematopoietic differentiation. Chromatin conformation analysis revealed that the MECOM regulated genes with cis-elements bound by CTCF underwent chromatin reorganization and became more highly looped as LT-HSCs underwent differentiation, suggesting opposing functions of MECOM and CTCF in the regulation of the MECOM gene network. In light of these findings, we performed tandem perturbation of MECOM and CTCF and demonstrated rescue of LT-HSC loss with the dual perturbation, thereby illuminating a key role for MECOM in constraining CTCF-dependent chromatin reorganization that occurs as HSCs undergo differentiation.Finally, based on the observation that elevated MECOM expression is associated with high-risk myeloid malignancies, we investigated the role of the MECOM-regulated HSC gene network in acute myeloid leukemias (AML). Across three independent AML datasets, we found that the MECOM regulated gene network had an independent and strong prognostic prediction ability that enabled risk stratification beyond currently used approaches, including a variety of molecular criteria and the previously described LSC17 signature. To validate these correlative observations, we performed CRISPR/Cas9 editing of MECOM in the MUTZ-3 AML cell line that is characterized by MECOM overexpression. We found that MECOM editing results in a loss of CD34 + leukemia progenitors and that the same transcriptional network that we identified in LT-HSCs is similarly altered upon MECOM perturbation in these AML cells.Collectively, we use the study of a rare experiment of nature due to MECOM haploinsufficiency resulting in neonatal aplastic anemia to illuminate a gene regulatory network necessary for HSC self-renewal and maintenance that is co-opted in high-risk forms of AML. DisclosuresRegev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees. Sankaran: Forma: Consultancy; Ensoma: Consultancy; Novartis: Consultancy; Cellarity: Consultancy; Branch Biosciences: Consultancy.

The Tetraspanin CD53 Protects Hematopoietic Stem Cells Following Cellular Stress through Induction of DREAM Complex-Mediated Quiescence

Maintenance of quiescence is necessary for optimal hematopoietic stem cell (HSC) function. The absence of fine-tuned cycling regulation of HSCs can result in impaired hematopoiesis, bone marrow (BM) failure, or malignant transformation. While various factors, including inflammatory cytokines and chemotherapy, have been identified to induce HSC cycling, the mechanisms regulating HSC return to quiescence are unclear.Herein, we profiled HSCs in response to a variety of inflammatory stressors (cytokines, toll-like receptor agonists, mobilizing agents, and chemotherapeutics) and found markedly upregulated expression of CD53 in both mouse and human HSCs, in some cases by as much as 70-fold higher expression over vehicle-treated controls. CD53 is a tetraspanin, a type of transmembrane protein involved in plasma membrane organization and regulation of processes such as cellular migration, adhesion, and signaling. CD53 has been shown to be asymmetrically segregated in HSCs, with CD53-enriched HSCs believed to be more stem-like; however, there is no current proposed mechanism to explain the association between CD53 and stem cell quality.To understand the role of CD53 in HSC quality, we generated a CD53 knockout mouse, and profiled HSC phenotype and function. Under homeostatic conditions, Cd53 -/- HSC number and frequency are normal as compared to wild type (WT) mice. However, we found Cd53 -/-HSCs to have significantly impaired function, particularly in response to inflammatory stimuli. Cd53 -/- BM failed to engraft as well as WT BM (45% chimerism vs 63%, p<0.05) in competitive transplants, and this deficit was exacerbated when G-CSF-mobilized HSCs from the spleen were transplanted (47% vs 77%, p<0.0001). Analysis of cycling status during and after G-CSF stimulation found that Cd53 -/- HSCs, particularly those that mobilize to the spleen, undergo cell division twice as frequently as WT HSCs (22% vs 10%, p<0.05). Treatment of WT and Cd53 -/- mice with serial doses of the chemotherapeutic 5-fluorouracil resulted in significantly accelerated death in Cd53 -/- mice (median survival 14 days vs 28 days, p<0.0001), supporting that loss of CD53 is associated with dysregulated HSC quiescence.Transcriptomic sequencing revealed significant upregulation of genes associated with cell cycling and division in G-CSF-treated Cd53 -/- HSCs compared to WT controls. Notably, these differentially expressed genes are targets of the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) complex, a newly described transcriptional regulator that represses cell cycling-associated genes, suggesting that CD53 promotes DREAM-mediated quiescence in stressed HSCs. We performed CUT&Tag profiling of Rbl2/p130, a DREAM complex subunit, in HSCs and found that loss of CD53 was associated with decreased complex binding (consistent with enhanced transcription of cell cycle genes). Proximity labeling studies demonstrated that CD53 interacts with cell cycle machinery proteins, including Rbl2/p130, and Western blots of sorted HSCs from Cd53 -/- and WT mice treated with G-CSF showed decreased expression of DREAM complex components (Rbl1/p107 and Rbl2/p130) in the absence of CD53. Finally, enforced activation of DREAM and HSC quiescence using the CDK4/6 inhibitor palbociclib rescued the Cd53 -/-HSC repopulating defect. Together, these data derive a novel mechanism whereby CD53 regulates HSC quiescence through regulating DREAM complex binding during stress-induced cycling. DisclosuresNo relevant conflicts of interest to declare.

Hormetic endoplasmic reticulum stress in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) possess the ability to regenerate over a lifetime in the face of extreme cellular proliferation and environmental stress. Yet, mechanisms that control the regenerative properties of HSCs remain elusive. ER stress has emerged as an important signaling event that supports HSC self-renewal and multipotency. The purpose of this review is to summarize the pathways implicating ER stress as cytoprotective in HSCs. Recent studies have shown multiple signaling cascades of the unfolded protein response (UPR) are persistently activated in healthy HSCs, suggesting that low-dose ER stress is a feature HSCs. Stress adaptation is a feature ascribed to cytoprotection and longevity of cells as well as organisms, in what is known as hormesis. However, assembling this information into useful knowledge to improve the therapeutic application of HSCs remains challenging and the upstream activators and downstream transcriptional programs induced by ER stress that are required in HSCs remain to be discovered. The maintenance of HSCs requires a dose-dependent simulation of ER stress responses that involves persistent, low-dose UPR. Unraveling the complexity of this signaling node may elucidate mechanisms related to regeneration of HSCs that can be harnessed to expand HSCs for cellular therapeutics ex vivo and transplantation in vivo.

CITED2 coordinates key hematopoietic regulatory pathways to maintain the HSC pool in both steady-state hematopoiesis and transplantation.

SummaryHematopoietic stem cells (HSCs) reside at the apex of the hematopoietic differentiation hierarchy and sustain multilineage hematopoiesis. Here, we show that the transcriptional regulator CITED2 is essential for life-long HSC maintenance. While hematopoietic-specific Cited2 deletion has a minor impact on steady-state hematopoiesis, Cited2-deficient HSCs are severely depleted in young mice and fail to expand upon aging. Moreover, although they home normally to the bone marrow, they fail to reconstitute hematopoiesis upon transplantation. Mechanistically, CITED2 is required for expression of key HSC regulators, including GATA2, MCL-1, and PTEN. Hematopoietic-specific expression of anti-apoptotic MCL-1 partially rescues the Cited2-deficient HSC pool and restores their reconstitution potential. To interrogate the Cited2→Pten pathway in HSCs, we generated Cited2;Pten compound heterozygous mice, which had a decreased number of HSCs that failed to reconstitute the HSC compartment. In addition, CITED2 represses multiple pathways whose elevated activity causes HSC exhaustion. Thus, CITED2 promotes pathways necessary for HSC maintenance and suppresses those detrimental to HSC integrity.

Disturbance of serotonergic regulation of hematopoietic stem cells (HSCS CD 34+) and blood mononuclear cells (BMCS) in patients with multiple sclerosis (MS)

Background and goals. Serotonin is involved in the mechanisms of regulation of the function of immunocompetent cells through the receptor apparatus. The aim of the study was to evaluate the expression capacity (EC) of BMCs and HSCs under the influence of serotonin - creatine sulfate at a dose of 10−4 M in the “in vitro”test system(TS). Violation of the receptor apparatus of cells serotonin led to a change in their quantitative registration.

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

Thrombopoietin from hepatocytes promotes hematopoietic stem cell regeneration after myeloablation.

The bone marrow niche plays critical roles in hematopoietic recovery and hematopoietic stem cell (HSC) regeneration after myeloablative stress. However, it is not clear whether systemic factors beyond the local niche are required for these essential processes in vivo. Thrombopoietin (THPO) is a key cytokine promoting hematopoietic rebound after myeloablation and its transcripts are expressed by multiple cellular sources. The upregulation of bone marrow-derived THPO has been proposed to be crucial for hematopoietic recovery and HSC regeneration after stress. Nonetheless, the cellular source of THPO in myeloablative stress has never been investigated genetically. We assessed the functional sources of THPO following two common myeloablative perturbations: 5-fluorouracil (5-FU) administration and irradiation. Using a Thpo translational reporter, we found that the liver but not the bone marrow is the major source of THPO protein after myeloablation. Mice with conditional Thpo deletion from osteoblasts and/or bone marrow stromal cells showed normal recovery of HSCs and hematopoiesis after myeloablation. In contrast, mice with conditional Thpo deletion from hepatocytes showed significant defects in HSC regeneration and hematopoietic rebound after myeloablation. Thus, systemic THPO from the liver is necessary for HSC regeneration and hematopoietic recovery in myeloablative stress conditions.

Dynamic Changes of the Bone Marrow Niche: Mesenchymal Stromal Cells and Their Progeny During Aging and Leukemia.

Mesenchymal stromal cells (MSCs) are a heterogenous cell population found in a wide range of tissues in the body, known for their nutrient-producing and immunomodulatory functions. In the bone marrow (BM), these MSCs are critical for the regulation of hematopoietic stem cells (HSC) that are responsible for daily blood production and functional immunity throughout an entire organism’s lifespan. Alongside other stromal cells, MSCs form a specialized microenvironment BM tissue called “niche” that tightly controls HSC self-renewal and differentiation. In addition, MSCs are crucial players in maintaining bone integrity and supply of hormonal nutrients due to their capacity to differentiate into osteoblasts and adipocytes which also contribute to cellular composition of the BM niche. However, MSCs are known to encompass a large heterogenous cell population that remains elusive and poorly defined. In this review, we focus on deciphering the BM-MSC biology through recent advances in single-cell identification of hierarchical subsets with distinct functionalities and transcriptional profiles. We also discuss the contribution of MSCs and their osteo-adipo progeny in modulating the complex direct cell-to-cell or indirect soluble factors-mediated interactions of the BM HSC niche during homeostasis, aging and myeloid malignancies. Lastly, we examine the therapeutic potential of MSCs for rejuvenation and anti-tumor remedy in clinical settings.

ANP32B-mediated repression of p53 contributes to maintenance of normal and CML stem cells

AbstractProper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell–specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and postinjury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with p53 and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from patients with chronic myelogenous leukemia (CML). Anp32b deletion enhances p53 transcriptional activity to impair LSC function in a murine CML model and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B and identify ANP32B as a potential therapeutic target in treating CML.

Regulatory Role of microRNAs Targeting the Transcription Co-Factor ZNF521 in Normal Tissues and Cancers.

Powerful bioinformatics tools have provided a wealth of novel miRNA–transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

Hmgb2 Is a Novel Transcriptional Regulator of Latexin in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Precisely regulated HSC pool maintenance and blood cell production is vital for organismal survival. We previously have identified a protein, latexin (Lxn), as a novel regulator of HSCs in mice, whose natural variation in the expression is inversely correlated with HSC population size. However, the molecular mechanisms involved in transcriptional regulation of Lxn in HSCs have not been clearly defined. By using DNA pull down and mass spectrometry, we identified several proteins that bind to the promoter region of Lxn. One top candidate is Hmgb2(High mobility group protein B2). ChIP Q-PCR assay and luciferase assay confirmed the binding of Hmgb2 to Lxn promoter with the potential role of transcriptional repressor. We also found that knock-down of Hmgb2 increases the endogenous Lxn expression at both mRNA and protein levels in hematopoietic cell line EML cells and primary HSC cells. As a result, HSC number was demonstrated decreased in vitro and in vivo due to decreased self-renewal and proliferation and increased apoptosis. This study, for the first time, revealed the transcriptional regulation of Lxn by Hmgb2. Disclosures No relevant conflicts of interest to declare.

A Cluster of Enhancer Modules Directs Differential Myc Expression Along the Normal and Leukemic Hematopoietic Stem Cell Hierarchies

Large enhancer regions, known as “super-enhancers”, have been described as critical regulatory units governing cell identity, but their actual contribution to stem cell-driven hierarchies remains poorly understood. The transcription factor MYC has critical roles in the regulation of hematopoietic stem cells (HSCs) and progenitors as well as in hematopoietic malignancies.Here we show that a region previously identified as a “super-enhancer”, located 1.7 Mb telomeric from Myc plays an essential role in regulating Myc expression throughout hematopoiesis. Deletion of this “super enhancer” in mice leads to a complete loss of Myc expression in HSC/progenitors, causing an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, similarly to the conditional deletion of the Myc gene in HSCs. This “Blood ENhancer Cluster” (BENC), which is functionally conserved in mice and humans, is comprised of distinct modules with selective activity and recruitment of such transcription factors as GFI1b, RUNX1 and MYB in different hematopoietic cell types. Analysis of mice carrying deletions of individual enhancer modules revealed a modular and combinatorial organization of BENC controlling Myc expression levels throughout most of the hematopoietic hierarchy. Additionally, we show that individual enhancer modules are activated at distinct stages during B cell development.In a malignant setting, we demonstrate that BENC is essential for the maintenance of MLL-AF9 leukemia in mice. Furthermore, in human acute myeloid leukemia, BENC modules show an increased accessibility in primary leukemic stem cells compared to blasts. The accessibility of the module directing Myc expression in normal HSCs correlates with Myc expression and a favorable patient outcome. Altogether, these data indicate a major role of BENC in regulating MYC levels across both normal hematopoietic and leukemic hierarchies. The composition of BENC may serve as a blueprint for other enhancer clusters operating across cellular hierarchies in other tissue types. DisclosuresNo relevant conflicts of interest to declare.

Fli1 Is Essential for the Maintenance of Hematopoietic Stem Cell Homeostasis and Function

The ETS family of transcription factors is known to play an essential role in hematopoietic and vascular development. One such factor that is widely expressed in all vascular beds and almost all hematopoietic lineages, extending from long term hematopoietic stem cells to terminally differentiated peripheral blood cells, is Fli1. Global deletion of Fli1 leads to embryonic lethality at E12.5 due to dramatic hemorrhaging caused by poor vascular integrity and platelet dysfunction. The role of Fli1 in adults has been explored in several different cells types ranging from megakaryocytes to B and T cells. It has also been implicated in transcriptional regulation of hematopoietic stem and progenitor cells through combinatorial analysis, however its exact contribution to stem cell maintenance and function remains unclear. We hypothesized that Fli1 plays a dual role in regulating both the seed and the soil - specifically hematopoietic stem cell (HSC) function in a cell autonomous manner as well as the niche required for nurturing these cells. In this study, we focused our attention on the cell autonomous function of Fli1. We found that global deletion of Fli1 leads to lethality as a result of complete peripheral blood failure in addition to aberrant vasculature. Using the cre-lox system and various transplantation strategies, we identify Fli1 as one of the critical regulators of adult hematopoietic stem cell function. Specific deletion of Fli1 in the hematopoietic compartment alone is sufficient to induce significant reduction in peripheral blood counts, accompanied by hemorrhage, resulting in lethality. On further inspection, Fli1-/- HSCs are unable to expand ex vivo or engraft in a competitive setting. Additionally, Fli1 is essential for hematopoietic reconstitution post radiation and its deletion abolishes the ability of stem cells to reconstitute the bone marrow and contribute to peripheral blood lineages. ChIP-seq analysis demonstrates Fli1 occupancy in almost all essential hematopoietic gene enhancers and promoters. By correlating Fli1-/- HSC RNA seq analysis with the ChIP occupancy data we can conclude that Fli1 is, in fact, one of the master regulators of the HSC transcriptional program essential to maintain the balance between self-renewal and differentiation. Taken together, our results indicate that Fli1 is required for maintenance of HSC homeostasis and function, making it one of the unique transcription factors to play a critical role in HSC function in adults as well as during development. DisclosuresNo relevant conflicts of interest to declare.

Heterodimerization of TPO and IFNγ Impairs Human Hematopoietic Stem/Progenitor Cell Signaling and Survival in Chronic Inflammation

Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling through JAK-STAT and other pathways. In recent clinical trials, eltrombopag, a small molecule c-MPL agonist, improved trilineage hematopoiesis in subjects with severe aplastic anemia (SAA). Paradoxically, TPO levels are already markedly elevated in these patients. To explain this paradox, we previously showed that IFNγ, a key proinflammatory cytokine implicated in the destruction of HSPCs in SAA, inhibited TPO signaling in human CD34+ HSPCs cultured in the presence of both cytokines. In contrast, eltrombopag could evade this inhibition in vitro, resulting in improved maintenance of progenitors in clonogenic CFU assays, and long-term repopulating cells in NSG transplantation models compared with TPO-containing cultures (Cheng et al., ASH abstract 2016). In this study, we sought to characterize the mechanisms by which eltrombopag evades IFNγ blockade of c-MPL signaling. Because activation of both c-MPL and IFNγ receptor by their respective ligands induces negative regulatory feedback mechanisms from the SOCS family, we first measured expression of SOCS proteins in CD34+ cells exposed to IFNγ and TPO or eltrombopag. SOCS expression was equally upregulated by TPO and eltrombopag in the presence of IFNγ, suggesting an alternative explanation for eltrombopag's ability to escape IFNγ-induced perturbation of TPO signaling. Because TPO and eltrombopag distinctively bind to the extracellular or juxtamembrane domain of c-MPL, respectively, we hypothesized that IFNγ may decrease binding affinity of TPO, but not eltrombopag, to c-MPL. We used microscale thermophoresis (MST) to assess the impact of IFNγ on TPO binding to its receptor. As previously reported, we showed that TPO and c-MPL interact via both a high affinity (KD, app<0.11 ± 0.04 nM) and a low affinity (KD, app=1100 ± 130 nM) binding site. In contrast, IFNγ had no interaction with c-MPL. Remarkably, addition of 100-molar excess IFNγ prevented binding of TPO to c-MPL at the low affinity site, but had no impact on the high affinity binding site (KD, app<0.20 ± 0.04 nM) (Panel A). To explain this result, we hypothesized that TPO and IFNγ may directly interact and form heteromeric complexes that hinder binding to c-MPL, whereas the non-peptide small molecule eltrombopag could conceivably evade that process. Strikingly, MST assays revealed a specific, one-site heterodimeric interaction between TPO and IFNγ (KD, app=540 ± 30 nM), while no heterodimer formation was observed between either TPO or IFNγ and other early-acting cytokines (SCF and Flt3L) (Panel B). These data suggest that TPO:IFNγ heterodimers may be responsible for the hindered TPO:c-MPL low-affinity interaction observed in the presence of IFNγ. To confirm this finding, we investigated whether TPO-induced c-MPL dimerization was interrupted in the presence of IFNγ. Using raster image correlation spectroscopy (RICS) to determine average diffusion coefficients of GFP-tagged c-MPL in live cells, we have preliminarily found that IFNγ negatively affects c-MPL dimerization in cells cultured with TPO, but not with eltrombopag. Further studies are underway. Taken together, our data provide a new and provocative paradigm to explain the observed negative impact of the proinflammatory cytokine IFNγ on human HSPC maintenance in bone marrow failure syndromes, and the ability of a small molecule (eltrombopag) to evade this inhibition. We propose that, under chronic inflammatory conditions, IFNγ specifically heterodimerizes with TPO, resulting in (i) occlusion of the low-affinity binding site of TPO to c-MPL, (ii) impaired receptor dimerization, (iii) perturbation of TPO-induced signaling pathways, and (iv) decreased survival of human HSPCs (Panel C). This new understanding could have far-reaching clinical implications for other disorders of chronic inflammation. [Display omitted] DisclosuresHuntsman:Novartis: Research Funding. Cheng:Novartis: Research Funding. Larochelle:Novartis: Research Funding; Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit.

Nfix  Promotes Survival of Immature Hematopoietic Cells Via Regulation of C-Mpl

Hematopoietic stem and progenitor cells (HSPC) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix -depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix -depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPCs, we overexpressed Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines and monitored the cultures for cell surface phenotype, growth, vector selection, and apoptosis. Ectopic expression of Nfix in primary mouse HSPCs had no significant effect on the expression of lineage markers compared to controls, but did appear to accelerate the loss of immunophenotypic LSK cells, as well as colony forming units (p = 0.023). Nfix -overexpressing HSPCs were extended in their ex vivo culture lifespan from about 20 days to 40 days, and were steadily selected for in culture. HSPCs overexpressing Nfix also displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls (p = 0.032). NFIX has been shown to potentially influence transcription of several known regulators of HSC biology. Here, we show that ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Consequently, Bcl-XL, an anti-apoptotic factor induced by STAT5, was also significantly upregulated in Nfix -overexpressing cells by two weeks of culture compared to controls (p = 0.0038). Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody significantly negated the anti-apoptotic effect of Nfix overexpression on cultured HSPCs (p = 0.045, p = 0.0098, respectively). Further, NFIX was capable of transcriptional activation of a proximal c-Mpl promoter fragment (p = 0.0056). In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo . DisclosuresNo relevant conflicts of interest to declare.

Role of SCF-Expressing Bone Marrow Populations in Hematopoietic and Leukemic Stem Cell Regulation

Stem cell factor (SCF), the ligand for the c-Kit receptor, stimulates growth of early hematopoietic stem/progenitors cells (HSCs) directly and/or by stimulating other cytokines. Using transgenic and driver/reporter mice, studies have shown that, in physiological conditions the Scf gene is expressed in bone marrow microenvironmental (BMM) cells, in particular endothelial and perivascular stroma. Deletion of exon 1 from the Scf gene in endothelial cells (using Tek-cre), perivascular cells (using Lepr-cre) and adipocytes (using adipoq-cre) significantly reduced the frequency and function of hematopoietic stem cells (HSCs) in young adult mice (Zhou et al. Nature cell biology. 2017; Ding et al. Nature. 2013). These studies have been useful in identifying the different Scf-expressing niche compartments from the BMM that are important for HSC maintenance. However, leukemic stem cell (LSC ) regulation by specific BMM cells in CML is poorly understood. In our study, the different Scf-expressing niche populations were identified using the Scf-GFP knock-in model (Scfgfp/+). CD45-TER119-CD31-CD140+ (PαS) and CD45-TER119-CD31-CD51+CD105+6C3+ (6C3) populations, representing mesenchymal progenitors and HSC-supporting stromal cells, respectively, showed the highest levels of Scf expression in normal mice, with endothelial cells also expressing SCF but at a lower level. We confirmed the role of Scf in normal hematopoiesis by showing that Scff/f - Ubc-cre mice generated by crossing Scff/f mice (LoxP sites flanking exon 1) with Ubc-cre mice (Cre mediated excision in widespread tissues following Tamoxifen administration) showed decreased stem/progenitor counts. To further evaluate the contribution of Scf-expressing populations towards regulation of normal HSC and LSCs, the Scff/f mice (LoxP sites flanking exon 1) were crossed with Bglap-cre (osteoblasts), Osx-cre (osteoprogenitors), Prx1-cre (mesenchymal progenitors) and Tek-cre (endothelial cells) mice. Deletion of Scf in the target populations was confirmed. We observed markedly reduced long-term HSC (LT-HSC; Lin-Sca-1+Kit+CD48-CD150+) numbers in the BM of 4-week old Scff/f-Prx1-cre mice, and to a lesser extent in Scff/f - Bglap-cre mice, and reduced short-term HSC (ST-HSC; Lin-Sca-1+Kit+CD48-CD150-). We observed markedly reduced ST-HSC numbers, and LT-HSC to a lesser extent, in the BM of 4-week old Scff/f - Osx-cre mice, supporting a role for these Scf -expressing populations in normal HSC maintenance in developing animals. In contrast to previous reports, we did not observe changes in stem/progenitor numbers in Tek-cre animals. To further evaluate effects of Scf-expressing niche populations on regulation of normal HSC and CML LSC, BM cells from wild-type (1x106 CD45.1 cells) and SCL-tTA-BCR-ABL mice (1x106 CD45.1/2) were transplanted into irradiated (8Gy) Scff/f Cre- or Cre+ knockout animals and followed for hematopoietic reconstitution. Scff/f-Prx1-cre animals showed decreased numbers of normal LT-HSC and ST-HSC in the BM compared to WT mice, but did not show changes in CML LSC numbers, although BM and peripheral blood (PB) myeloid counts were reduced. Scff/f - Osx-cre mice showed decreased numbers of ST-HSC, but no changes in changes in normal HSC or CML LSC were seen. Scff/f-Bglap-cre mice did not show significant reduction in normal and CML LT-HSC in BM. Collectively, our results show that Scf -expressing mesenchymal stem cells play an important role in normal LT-HSC maintenance, and osteoprogenitors in normal ST-HSC maintenance. In contrast, CML LSCs are not dependent on SCF expression on the different mesenchymal populations studied here. Therefore, normal and leukemic stem cells appear to be differentially regulated by SCF-expressing BMM populations. Further studies to better define the Scf -expressing populations that support CML LSCs, and the effects of SCF-expressing BMM populations on LSC response to tyrosine kinase inhibitors are underway. DisclosuresNo relevant conflicts of interest to declare.

Potential Role of Ubiquitin E3 Ligase, FBXO9, As a Tumor Suppressor in AML

Hematopoietic stem cells (HSCs) give rise to all cells of the blood. The balance between HSC self-renewal and differentiation is critical to maintaining hematopoiesis throughout the life of the organism, and disruptions to this balance often result in malignancies, including acute myeloid leukemia (AML). Multiple transcription factor networks are known to be important regulators of HSCs and, when mutated, can lead to leukemia development. More recently, protein degradation by the ubiquitin proteasome system (UPS) has been shown to regulate HSC self-renewal, differentiation, and survival. The role of this pathway in leukemogenesis is less clear.We recently performed a screen in embryonic stem cells to identify members of the UPS involved in cell fate decisions. From this, we identified the Ubiquitin E3 ligase FBXO9, which is a substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex. Interestingly, FBXO9 is highly expressed in hematopoietic stem and progenitor cells, but is decreased in patients with AML including patients with Inv(16) chromosomal abnormality. In order to study FBXO9 in normal and malignant hematopoiesis, we utilized a novel method using CRISPR/Cas9 to generate a conditional knockout model in collaboration with the Mouse Transgenic Core at UNMC. We bred our mice to transgenic mice expressing Cre Recombinase from the Mx1 promoter, which allows for PolyIC inducible expression of Cre in all hematopoietic cells including HSC. Our preliminary data shows alterations in hematopoiesis, including less competitive repopulation capability of the HSC in vivo and decreased differentiation potential of the hematopoietic progenitor population (HPC). To test if Fbxo9 plays a role in leukemia development, we crossed mice with the conditional allele of Fbxo9 with a knockin mouse model of AML that expresses the leukemia fusion gene Cbfb-MYH11 . In a small cohort of mice, we found that deletion of Fbxo9 in the context of Cbfb-MYH11 caused markedly accelerated leukemia development with >50% myeloid blasts in the peripheral blood by week 6. These findings suggest that the ubiquitin E3 ligase, Fbxo9, is a novel tumor suppressor and can regulate self-renewal, differentiation, and malignant transformation. In addition The dynamic reversibility of the ubiquitin modification (by kinases, phosphatases, E3 ligases, and de-ubiquitinases) and the recent development of both general proteasome inhibitors as well as specific antagonists of ligase function suggest that manipulation of proteins by ubiquitin E3 ligases could provide potential targets for drug discovery and stem cell therapy. DisclosuresNo relevant conflicts of interest to declare.

Extracellular DEK Regulates Hematopoiesis through CXCR2-Mediated Intracellular Signaling That Does Not Require Its Translocation into the Nucleus or Binding to DNA

DEK is a highly conserved, ubiquitously expressed and structurally unique DNA-binding nuclear protein involved in transcription, DNA replication, and DNA repair implicating a role for DEK in gene regulation. Interestingly, hematopoietic cells, following stimulation with inflammatory mediators, can actively secrete DEK, which can then be taken up by cells via binding to heparan sulfate proteoglycans (HSPGs), translocating to the nucleus, and mediating its normal function of gene regulation. Previous work from our group indicates that both endogenous, intracellular DEK and secreted, extracellular DEK regulates normal hematopoiesis by decreasing numbers of hematopoietic progenitor cells (HPCs) and increasing numbers of hematopoietic stem cells (HSCs) suggesting that DEK plays a role in regulating HSC/HPC function and/or cell fate. Extracellular DEK-mediated regulation of hematopoiesis occurs in a HSPG-, CXCR2-, Gai protein-dependent manner. Neutralizing these pathways inhibits extracellular DEK regulation of HSCs and HPCs. However, what was not known is whether the HSPG-dependent internalization of DEK is required for the extracellular DEK-mediated regulation of hematopoiesis as HSPGs are also required for maximum binding efficiency of ELR-CXC chemokines to CXCR2. To address this unknown, two mutant forms of recombinant (r)DEK were utilized: DEK that lacks its nuclear translocation sequence (NTS) and DEK that lacks its DNA-binding motif (DBM). First we examined whether NTS- or DMB-rDEK could alter HPC or HSC numbers in vitro / ex vivo . We had previously shown that C57Bl/6 bone marrow (BM) cells when in culture with rDEK inhibited HPC colony formation in a dose-dependent manner, with the minimum dose of WT rDEK required for inhibition being 12.5 nM (p=0.002). Both NTS- and DMB-rDEK also inhibited HPC colony formation in a dose-dependent manner starting at 12.5 nM (p=0.03 and p=0.02 respectively) suggesting that neither the ability of DEK to get into the nucleus or bind to DNA is required to regulate HPC numbers in BM. To examine whether NTS- or DMB-rDEK can still enhance HSC numbers, ex vivo HSC expansion assays were performed. C57Bl/6 lineage negative BM cells were cultured in SCF, TPO, and Flt3L with vehicle control, WT-rDEK, NTS-rDEK, or DMB-rDEK then examined for HSC expansion on days 4 and 7. On both days, WT-, NTS-rDEK and DMB-rDEK significantly expanded long-term HSC numbers when compared to vehicle control, suggesting that like DEK-mediated suppression of HPCs, DEK-mediated expansion of HSCs does not require DEK to translocate to the nucleus or bind to DNA. To examine whether extracellular NTS- and DMB-rDEK can still mediate one of DEK's known nuclear functions, heterochromatin stability, DEK-/- lineage negative BM cells were incubated for 12 hours with WT rDEK, NTS-rDEK or DMB-rDEK then examined for H3K9 trimethylation in HSCs and HPCs by ImageStream flow cytometry. Neither NTS- or DMB-rDEK were able to enhance the trimethylation of H3K9 in DEK-/- HSCs or HPCs. However, incubating DEK-/- lineage negative BM with WT rDEK, increased H3K9me3 in the nucleus of the cells by ~26%. When utilizing WT BM, incubating cells for 12 hours with WT, NTS- or DMB-rDEK did not result in changes in H3K9Me3 that was detectable by ImageStream technology suggesting that extracellular DEK-mediated changes in chromatin remodeling in HSCs and HPCs only occurs when the cells lack endogenous DEK. Co-culturing C57Bl/6 BM cells with WT rDEK mediated AKT and ERK1/2 phosphorylation in HSCs and ERK1/2 in HPCs which is blocked when BM cells are pretreated with a neutralizing antibody against CXCR2 or when preteated with heparin neutralizing HSPGs. Both NTS- and DMB-rDEK were able to mediate similar activation of AKT and ERK1/2 in HSCs and HPCs as WT rDEK but not when the BM was pretreated with anti-CXCR2 antibody or heparin. Taken together, these data suggest that extracellular DEK functions to regulate HSC and HPC numbers/function by binding to CXCR2 to mediate intracellular signaling events in C57Bl/6 BM cells. The fact that HSPGs are required for extracellular DEK's effect, but DEK does not have to get into the nucleus and bind DNA, also suggests that internalization of DEK is not required to regulate hematopoiesis in cells with normal levels of endogenous DEK. Therefore, like the ELR-CXC chemokines, DEK requires HSPGs for its CXCR2-mediated signaling processes in both HSCs and HPCs. DisclosuresNo relevant conflicts of interest to declare.

Gain of Function Mutant p53 Drives Clonal Hematopoiesis and the Pathogenesis of Myelodysplastic Syndromes

Clonal hematopoiesis increases with age, where a single mutant hematopoietic stem or progenitor cell (HSPC) contributes to a significant, measurable clonal proportion of mature blood lineages. Evolution of mutant clonal hematopoiesis with age predisposes the elderly to myelodysplastic syndromes (MDS) and other aging-associated diseases, suggesting that mutations identified in HSPCs contribute to disease development. Previous studies using whole exome sequencing of blood DNAs identified a high frequency of TP53 mutations in aged healthy individuals . TP53 mutations are also present in 10% of MDS cases and correlate with worse clinical outcomes. However, the role of TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Ample data indicate that mutant p53 proteins not only lose their tumor suppressive functions, but also gain new abilities that promote tumorigenesis. Here, we report that specific gain-of-function (GOF) mutant p53 proteins drive the development of pre-leukemic hematopoietic stem cells (HSCs), predisposing aged mutant p53 mice to MDS development.Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. We found that GOF mutant p53 (p53R248W) enhances the competitive advantage of HSCs without affecting terminal differentiation in steady state. Further, we observed that p53R248W mutant promotes clonal expansion of HSCs following genotoxic stresses. RNA-seq analysis revealed that HSC and leukemia stem cell signatures were significantly enriched in mutantHSCs compared to wild-type HSCs. Thus, HSCs expressing GOF mutant p53 recapitulate several key features of pre-leukemic HSCs. GOF p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. We discovered that GOF mutant p53 proteins, including p53R248W, p53R273H, and p53R175H, but not wild-type p53, interact with histone methyltransferase EZH2 and enhance H3K27me3 in HSCs. EZH2, a catalytic component of the Polycomb repressive complex 2 (PRC2), trimethylates histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes. Indeed, we found that the expression of HSC regulators, including Elf4 and Gadd45g, was significantly downregulated in mutant HSCs compared to wild-type HSCs. ChIP-seq analysis show enrichment of H3K27me3 in the promoter regions of Elf4 and Gadd45g in mutant HSCs. Importantly, we discovered that EZH2 inhibitor treatment decreased the replating potential of mutantbone marrow cells to wild-type cell level. Thus, we demonstrated that GOF mutant p53 drives the development of pre-leukemic HSCs through enhancing EZH2 activity.Given that most homozygous p53-/- and p53R248W/R248Wmice develop spontaneous tumors, including lymphoma, thymoma, and sarcoma, and die within 3 to 6 months after birth, it was not clear whether heterozygous mutant p53 mice (p53R248W/+) would develop myeloid malignancies during aging. To evaluate the role of GOF mutant p53 in the pathogenesis of myeloid malignancies, we maintained p53+/+ and p53R248W/+ mice for more than a year and monitored their survival and tumor development. While p53R248W/+ mice show extended life span compared to p53-/- and p53R248W/R248W mice, most of these mice died within 16-18 months after birth. During the observation period, approximately 30 % of heterozygous mutant p53mice developed MDS based upon pathological analysis of BM, spleen, and peripheral blood smear. Histological assessment revealed hypercellular BM in some aged p53R248W/+ mice. BM of some aged p53R248W/+ mice show neutrophil, erythroid precursor and megakaryocyte dysplasia. We further demonstrated that mutant p53 driven-MDS is transplantable. Thus, we provided evidence that GOF mutant p53 drives MDS development during aging.In summary, we have identified a critical role for GOF mutant p53 in clonal hematopoiesis and the pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.